Assessing viral metagenomics for the diagnosis of acute undifferentiated fever in returned travellers: a multicenter cohort study.

BACKGROUND: Up to 45% of febrile returning travellers remain undiagnosed after a thorough diagnostic work-up, even at referral centres. Although metagenomic next-generation sequencing (mNGS) has emerged as a promising tool, evidence of its usefulness in imported fever is very limited. METHODS: Travellers returning with fever were prospectively recruited in three referral clinics from November 2017 to November 2019. Unbiased mNGS optimised for virus detection was performed on serum samples of participants with acute undifferentiated febrile illness (AUFI), and results were compared to those obtained by reference diagnostic methods (RDM). RESULTS: Among 507 returned febrile travellers, 433(85.4%) presented with AUFI. Dengue virus (n = 86) and Plasmodium spp. (n = 83) were the most common causes of fever. 103/433(23.8%) AUFI remained undiagnosed at the end of the follow-up.Metagenomic next-generation sequencing unveiled potentially pathogenic microorganisms in 196/433(38.7%) AUFI. mNGS identifications were more common in patients with a shorter duration of fever (42.3% in ≤5 days vs 28.7% in >5 days, P = 0.005). Potential causes of fever were revealed in 25/103(24.2%) undiagnosed AUFI and 5/23(21.7%) travellers with severe undiagnosed AUFI. Missed severe aetiologies included eight bacterial identifications and one co-infection of B19 parvovirus and Aspergillus spp.Additional identifications indicating possible co-infections occurred in 29/316(9.2%) travellers with AUFI, and in 11/128(8.6%) travellers with severe AUFI, who had received a diagnosis through RDM. The most common co-infections detected in severe AUFI were caused by Gram-negative bacteria. Serum mNGS was unable to detect >50% of infectious diagnoses achieved by RDM and also yielded 607 non-pathogenic identifications. DISCUSSION: mNGS of serum can be a valuable diagnostic tool for selected travellers with undiagnosed AUFI or severe disease in addition to reference diagnostic techniques, especially during the first days of symptoms. Nevertheless, mNGS results interpretation presents a great challenge. Further studies evaluating the performance of mNGS using different sample types and protocols tailored to non-viral agents are needed.

Failure of artemether-lumefantrine therapy in travellers returning to Belgium with Plasmodium falciparum malaria: an observational case series with genomic analysis.

BACKGROUND: Failure of artemisinin-based combination therapy is increasingly reported in patients with Plasmodium falciparum malaria in sub-Saharan Africa. We aimed to describe the clinical and genomic characteristics of recent cases of P. falciparum malaria failing artemether-lumefantrine in Belgium. METHODS: Travel-related cases of malaria confirmed at the national reference laboratory of the Institute of Tropical Medicine, Antwerp, Belgium, were reviewed. All cases for which attending clinicians reported persistence (beyond Day 3 post-treatment initiation, i.e. early failure) or recrudescence (from Day 7 to 42, i.e. late failure) of P. falciparum parasites despite adequate drug intake were analysed. Both initial and persistent/recurrent samples were submitted to next generation sequencing to investigate resistance-conferring mutations. RESULTS: From July 2022 to June 2023, eight P. falciparum cases of failure with artemether-lumefantrine therapy were reported (early failure = 1; late failure = 7). All travellers were returning from sub-Saharan Africa, most (6/8) after a trip to visit friends and relatives. PfKelch13 (PF3D7_1343700) mutations associated with resistance to artemisinin were found in two travellers returning from East Africa, including the validated marker R561H in the patient with early failure and the candidate marker A675V in a patient with late failure. Additional mutations were detected that could contribute to decreased susceptibility to artemisinin in another three cases, lumefantrine in six cases and proguanil in all eight participants. Various regimens were used to treat the persistent/recrudescent cases, with favourable outcome. CONCLUSION: Within a 12-month period, we investigated eight travellers returning from sub-Saharan Africa with P. falciparum malaria and in whom artemether-lumefantrine failure was documented. Mutations conferring resistance to antimalarials were found in all analysed blood samples, especially against lumefantrine and proguanil, but also artemisinin. There is a pressing need for systematic genomic surveillance of resistance to antimalarials in international travellers with P. falciparum malaria, especially those experiencing treatment failure.

No Varicella Zoster Virus Infection among Mpox Cases in Antwerp, Belgium.

Several studies in tropical settings have reported that ∼20% of patients infected with Monkeypox virus (MPXV) also tested polymerase chain reaction (PCR) positive for Varicella zoster virus (VZV). Researchers have hypothesized that VZV infection predisposes to monkeypox (mpox), or vice versa, or that MPXV triggers the reactivation of VZV. We tested samples for VZV from a cohort of patients infected with clade IIb MPXV diagnosed between May 23 and October 14, 2022 in Antwerp, Belgium. Leftover DNA extracts of skin lesion samples from 108 mpox patients were tested with in-house PCR for VZV. No VZV infections were found. The absence of concurrent VZV-MPXV infections in our cohort indicates that VZV did not cocirculate in the population at risk for MPXV during the Belgian 2022 outbreak, but also that MPXV does not commonly trigger reactivation of latent VZV in adult men.

Detection of mpox virus in ambient air in a sexual health clinic.

Although transmitted mainly through direct (sexual) contact, mpox virus (MPXV) can be detected in ambient air. We explored the use of air sampling for diagnosis or (genomic) surveillance of mpox in a sexual health clinic. For six out of six patients who were infected with MPXV, all four of our ambient air PCR tests were positive. For 14 uninfected patients, PCR was positive in three ambient air samples, albeit with higher cycle threshold (Ct) values. Genomic sequencing of samples from two positive patients showed matching sequences between air and clinical samples.

Microscopic Detection of Intestinal Sarcocystis Infection Diagnosed in International Travelers at the Institute of Tropical Medicine, Antwerp, Belgium, from 2001 to 2020.

Although a stay in tropical regions is considered a risk factor for acquiring Sarcocystis infection, to date intestinal sarcocystosis has never been described in returning travelers. We did a retrospective cross-sectional study, retrieving all Sarcocystis spp. microscopy-positive stool results of individuals who attended the travel clinic of the Institute of Tropical Medicine, Antwerp in the period from 2001 to 2020. We reviewed the medical records and report on the epidemiology and clinical features of intestinal sarcocystosis in international travelers. In 57 (0.09%) of 60,006 stool samples, oocysts or sporocysts of Sarcocystis spp. were found, often together with other intestinal infections. Twenty-two (37%) individuals were asymptomatic, 17 (30%) had intestinal ± extraintestinal symptoms, and 18 (32%) had extraintestinal symptoms only. Only one traveler had symptoms suggestive of acute gastrointestinal sarcocystosis without an alternative diagnosis. Intestinal Sarcocystis infection predominated in male travelers. At least 10 travelers most likely acquired intestinal Sarcocystis in Africa, where it was never described before. In a national reference travel clinic in Europe, the presence of intestinal Sarcocystis oocysts is a rare finding, predominant in male travelers. Infection with this parasite infrequently leads to suggestive clinical manifestations such as acute gastrointestinal symptoms. Our data strongly suggest that Sarcocystis can be acquired throughout tropical areas, including Africa.

Presymptomatic viral shedding in high-risk mpox contacts: A prospective cohort study.

The risk of infection after exposure to clade IIb mpox virus (MPXV) is unknown, and potential presymptomatic shedding of MPXV remains to be demonstrated. High-risk contacts of mpox patients were followed-up in a prospective longitudinal cohort study. Individuals reporting sexual contact, >15 min skin-to-skin contact, or living in the same household with an mpox patient were recruited in a sexual health clinic in Antwerp, Belgium. Participants kept a symptom diary, performed daily self-sampling (anorectal, genital, and saliva), and presented for weekly clinic visits for physical examination and sampling (blood and oropharyngeal). Samples were tested for MPXV by PCR. Between June 24 and July 31, 2022, 25 contacts were included, of which 12/18 (66.0%) sexual and 1/7 (14.0%) nonsexual contacts showed evidence of infection by MPXV-PCR. Six cases had typical mpox symptoms. Viral DNA was detected as early as 4 days before symptom onset in 5 of them. In 3 of these cases, replication-competent virus was demonstrated in the presymptomatic phase. These findings confirm the existence of presymptomatic shedding of replication-competent MPXV and emphasize the high risk of transmission during sexual contact. Sexual contacts of mpox cases should abstain from sex during the incubation period, irrespective of symptoms.

Phylogeographic analysis of dengue virus serotype 1 and cosmopolitan serotype 2 in Africa.

OBJECTIVES: The origin and spread of dengue virus (DENV) circulating in Africa remain poorly characterized, with African sequences representing <1% of global sequence data. METHODS: Whole genome sequencing was performed on serum samples (n = 29) from an undifferentiated fever study in 2016 in the Democratic Republic of Congo (DRC), and from febrile travelers returning from Africa. The evolutionary history of the newly acquired African DENV-1 (n = 1) and cosmopolitan genotype DENV-2 (n = 18) genomes was reconstructed using a phylogeographic, time-scaled Bayesian analysis on a curated DENV panel including all known African sequences. RESULTS: A minimum of 10 and eight introductions could be identified into Africa for DENV-1 and cosmopolitan DENV-2, respectively, almost all originating from Asia. Three introductions were previously unknown. The currently circulating virus comprises mainly the recently introduced clades and one long-established African clade. Robust geographical clustering suggests limited spread of DENV after each introduction. Our data identified the DRC as the source of the 2018 Angolan DENV-2 epidemic, and similarly, the 2013 Angolan DENV-1 outbreak as the origin of our DRC study. CONCLUSION: Active genomic surveillance of DENV in Africa at the portals of entry might help early outbreak response and limit sero- and genotype spread and human disease burden.

Characteristics of confirmed mpox cases among clinical suspects: A prospective single-centre study in Belgium during the 2022 outbreak.

Background: The presentation of mpox clade IIb during the 2022 outbreak overlaps with a range of other diseases. Understanding the factors associated with mpox is important for clinical decision making. Methods: We described the characteristics of mpox patients who sought care at Belgian sexual health clinic. Furthermore we compared their characteristics to those of patients with a clinical suspicion of mpox but who tested negative on polymerase chain reaction. Results: Between May 23 and September 20, 2022, 155 patients were diagnosed with mpox, and 51 patients with suspected symptoms tested negative. All mpox patients self-identified as men and 148/155 (95.5%) as gay or bisexual MSM. Systemic symptoms were present in 116/155 (74.8%) patients. All but 10 patients (145/155, 93.5%) presented with skin lesions. Other manifestations were lymphadenopathy (72/155, 46.5%), proctitis (50/155, 32.3%), urethritis (12/155, 7.7%), tonsillitis (2/155, 1.3%). Complications involved bacterial skin infection (13/155, 8.4%) and penile oedema with or without paraphimosis (4/155, 2.6%). In multivariable logistic regression models, the presence of lymphadenopathy (OR 3.79 95% CI 1.44-11.49), skin lesions (OR 4.35 95% CI 1.15-17.57) and proctitis (OR 9.41 95% CI 2.72-47.07) were associated with the diagnosis of mpox. There were no associations with age, HIV status, childhood smallpox vaccination, number of sexual partners and international travel. Conclusions: The presence of proctitis, lymphadenopathies and skin lesions should increase clinical suspicion of mpox in patients with compatible symptoms.

Evaluation of the AIX1000 Automated Rapid Plasma Reagin Assay in a High-Prevalence Setting.

BACKGROUND: Manually performed nontreponemal assays, such as rapid plasma reagin (RPR), are labor intensive and time consuming. Recently, commercial automated RPR assays gained attention. The aim of this study was to compare the qualitative and quantitative performance of the AIX1000 (RPR-A; Gold Standard Diagnostics) to a manual RPR test (RPR-M; Becton Dickinson Macrovue) within a high-prevalence setting. METHODS: A retrospective panel of 223 samples was selected for comparison between RPR-A and RPR-M, including 24 samples from patients with known syphilis stages and 57 samples from 11 patients in follow-up. In addition, 127 samples obtained during routine syphilis diagnosis with RPR-M were analyzed prospectively with AIX1000. RESULTS: Overall qualitative concordance (percent agreement) between both assays was 92.0% in the retrospective and 89.0% in the prospective panel. Of 32 discordances, 28 were explained by a treated syphilis infection still positive in one assay and already negative in the other. One sample was false positive with RPR-A, 1 infection remained undetected by RPR-M, and 2 remained undetected by RPR-A. A hook effect was apparent on the AIX1000 at RPR-A titers from 1:32 onward; however, no infections were missed. Accepting a ±1 titer difference, quantitative concordance between both assays reached 73.1% and 98.4% for the retrospective and prospective panels, respectively, with an upper limit of reactivity for RPR-A at 1:256. CONCLUSIONS: The AIX1000 showed a similar performance to Macrovue RPR with the exception of a negative deviation for high-titer samples. Within the reverse algorithm used in our high-prevalence setting, AIX1000's main advantage is automation.

Clinical evaluation of BioFire® multiplex-PCR panel for acute undifferentiated febrile illnesses in travellers: a prospective multicentre study.

BACKGROUND: Identifying the causes of Acute Undifferentiated Febrile Illness (AUFI) is key to improve the management of returning travellers with fever. We evaluated a BioFire®FilmArray® prototype panel of multiplex nucleic acid amplification tests (NAAT) targeting different relevant pathogens in travellers returning with fever. METHODS: Prospective, multicentre study to evaluate a prototype panel in whole blood samples of adult international travellers presenting with AUFI in three European travel Clinics/Hospitals (November 2017-November 2019). We evaluated 15 target analytes: Plasmodium spp., Plasmodium falciparum, Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, chikungunya virus, dengue virus, Zika virus, Anaplasma phagocytophilum, Borrelia spp., Leptospira spp., Orientia tsutsugamushi, Rickettsia spp. and Salmonella spp. Results were compared with composite reference standards (CRSs) for each target infection, including direct methods [smear microscopy, rapid diagnostic test (RDT), reference NAAT and blood cultures] and indirect methods (paired serology). FINDINGS: Among 455 travellers with AUFI, 229 target infections were diagnosed; the prototype panel detected 143 (overall sensitivity and specificity of 62.5 and 99.8%, respectively). The panel identified all Plasmodium infections (n = 82). Sensitivity for dengue (n = 71) was 92.9, 80.8 and 68.5% compared with RDT, NAAT and CRS, respectively. Compared with direct methods and CRS, respectively, the prototype panel detected 4/4 and 4/6 chikungunya, 2/2 and 4/29 Leptospira spp., 1/1 and 1/6 O. tsutsugamushi and 2/2 and 2/55 Rickettsia spp., but 0/2 and 0/10 Zika, 0/1 and 0/11 A. phagocytophylum and 0/3 Borrelia spp. diagnosed by serology and only 1/7 Salmonella spp. diagnosed by blood cultures. 77/86 (89.5%) infections not detected by the panel were diagnosed by serology. INTERPRETATION: The prototype panel allowed rapid and reliable diagnosis for malaria, dengue and chikungunya. Further improvements are needed to improve its sensitivity for Zika and important travel-related bacterial infections.

Validation of a Reporter Cell Line for Flavivirus Inhibition Assays.

Here, we report the validation of a new reporter cell line, Hec1a-IFNB-Luc, for use in inhibition studies of various flaviviruses relevant to human pathology. The reporter system allows the detection of viral replication after luciferase gene activation driven by an interferon beta (IFN-ß) promoter. We found the reporter cell line to be highly responsive to all 10 flaviviruses tested, including the 4 dengue virus serotypes. The applicability of the Hec1a-IFNB-Luc reporter cell line for serodiagnostic purposes in neutralizing antibody assays was confirmed by comparison of its sensitivity and specificity to those of "gold-standard," clinically applied, cytopathic effect-based assays, showing comparable performances. The reporter cell line used for the assessment of viral inhibition by small-molecule antiviral compounds was also confirmed, and the sensitivity of the Hec1a-IFNB-Luc reporter cell line was compared to those from published data reporting on the activity of the antivirals in various other assays, indicating that the Hec1a-IFNB-Luc reporter cell line allowed the determination of the inhibitory capacity at least as sensitive as alternative assays. By measuring luciferase activity as a proxy for viral replication, the reporter cell line allows early detection, reducing the time to results from often 5 to 7 days to 3 days, without the need for optical inspection of cytopathic effects, which often differ between viruses and cell lines, streamlining the development of flavivirus assays. IMPORTANCE The Hec1a-IFNB-Luc reporter cell line allows the detection of all 10 flaviviruses tested, including the 4 dengue virus serotypes. Its use for serodiagnostic purposes, measuring neutralizing antibody activity in sera, and the assessment of the antiviral activities of small-molecule compounds was confirmed, and it was found to be comparable to clinically applied assays. The Hec1a-IFNB-Luc reporter cell line allows the rapid and quantitative determination of antiviral effects on multiple human pathological flaviviruses using a single protocol.

Persistent morbidity in Clade IIb mpox patients: interim results of a long-term follow-up study, Belgium, June to November 2022.

While mpox was well characterised during the 2022 global Clade IIb outbreak, little is known about persistent morbidity. We present interim results of a prospective cohort study of 95 mpox patients assessed 3-20 weeks post-symptom onset. Two-thirds of participants had residual morbidity, including 25 with persistent anorectal and 18 with genital symptoms. Loss of physical fitness, new-onset/worsened fatigue and mental health problems were reported in 36, 19 and 11 patients, respectively. These findings require attention by healthcare providers.

Alternative sampling specimens for the molecular detection of mpox (formerly monkeypox) virus.

BACKGROUND: Mpox (formerly monkeypox) is a viral disease caused by the mpox virus (MPXV), endemic in Central and West Africa and currently causing a global outbreak of international concern. Much remains unknown about sample types most suited for mpox laboratory diagnosis. While it is established that high viral loads can be found in active skin lesions (currently the recommended mpox laboratory confirmation specimen type), WHO mpox testing guidelines encourage the use of oropharyngeal swabs as an additional sample type for mpox diagnosis and suggest investigating the value of other specimens like blood samples. OBJECTIVE: In this study, we verified the value of select alternative specimen types for mpox laboratory confirmation. METHODS: We included 25 patients with MPXV-confirmed skin lesions to compare diagnostic sensitivity of MPXV PCR testing on EDTA plasma and two upper respiratory specimens: oropharyngeal swabs and saliva. RESULTS: In our patient cohort with MPXV-confirmed skin lesions, diagnostic sensitivity of MPXV PCR was 80% in EDTA plasma, 64% in oropharyngeal swabs, and 88% in saliva. MPXV viral loads were significantly higher in saliva compared to oropharyngeal swabs and EDTA plasma. DISCUSSION: The WHO recommendation to collect oropharyngeal swabs as an additional specimen for mpox diagnosis might need to be revised to include saliva wherever feasible. We suggest investigating saliva as a diagnostic specimen in the absence of active skin lesions or during the phase preceding skin manifestations. Moreover, the relatively high MPXV DNA content of saliva warrants elucidating its potential role in disease transmission.

Doxycycline responding illnesses in returning travellers with undifferentiated non-malaria fever: a European multicentre prospective cohort study.

BACKGROUND: Diagnosis of undifferentiated non-malaria fevers (NMF) in returning travellers is a great challenge. Currently, there is no consensus about the use of empirical antibiotics in returning travellers with undifferentiated NMF. Although studies in endemic areas showed that a wide range of pathogens implicated in undifferentiated NMF are treatable with doxycycline, the role of doxycycline in returning travellers with fever still has to be explored. METHODS: Prospective European multicentre cohort study of febrile international travellers (November 2017-November 2019). Immunological and molecular diagnostic techniques for doxycycline responding illnesses (DRI) agents such as Anaplasma phagocytophilum, spotted fever group Rickettsia spp., typhus group Rickettsia spp., Coxiella burnetii, Bartonella spp., Orientia tsutsugamushi, Borrelia miyamotoi, Borrelia recurrentis and Leptospira spp. were systematically performed in all patients with undifferentiated NMF. We estimated the prevalence and predictive factors of DRI in returning travellers with undifferentiated NMF. RESULTS: Among 347 travellers with undifferentiated NMF, 106 (30·5%) were finally diagnosed with DRI. Only 57 (53·8%) of the 106 DRI infections were diagnosed by the standard of care. The main causes of DRI were: 55 (51·9%) Rickettsia spp., 16 (15·1%) C. burnetii; 15 (14·2%) Bartonella spp.; 13 (12·3%) Leptospira spp. and 10 (9·5%) A. phagocytophilum. The only predictive factor associated with DRI was presenting an eschar (aOR 39·52, 95%CI 4·85-322·18). Features of dengue such as retro-orbital pain (aOR 0·40, 95%CI 0·21-0·76) and neutropenia (aOR 0·41, 95%CI 0·21-0·79) were negatively associated with DRI. CONCLUSIONS: Although DRI are responsible for 30% of undifferentiated NMF cases in travellers, those are seldom recognized during the first clinical encounter. Empirical treatment with doxycycline should be considered in returning travellers with undifferentiated fever and negative tests for malaria and dengue, particularly when presenting severe illness, predictive factors for rickettsiosis or no features of dengue.

Accuracy of C-reactive Protein and Procalcitonin for Diagnosing Bacterial Infections Among Subjects With Persistent Fever in the Tropics.

Background: In low-resource settings, inflammatory biomarkers can help identify patients with acute febrile illness who do not require antibiotics. Their use has not been studied in persistent fever (defined as fever lasting for ≥7 days at presentation). Methods: C-reactive protein (CRP) and procalcitonin (PCT) levels were measured in stored serum samples of patients with persistent fever prospectively enrolled in Cambodia, the Democratic Republic of Congo, Nepal, and Sudan. Diagnostic accuracy was assessed for identifying all bacterial infections and the subcategory of severe infections judged to require immediate antibiotics. Results: Among 1838 participants, CRP and PCT levels were determined in 1777 (96.7%) and 1711 (93.1%) samples, respectively, while white blood cell (WBC) count was available for 1762 (95.9%). Areas under the receiver operating characteristic curve for bacterial infections were higher for CRP (0.669) and WBC count (0.651) as compared with PCT (0.600; P <.001). Sensitivity for overall and severe bacterial infections was 76.3% (469/615) and 88.2% (194/220) for CRP >10 mg/L, 62.4% (380/609) and 76.8% (169/220) for PCT >0.1 µg/L, and 30.5% (184/604) and 43.7% (94/215) for WBC >11 000/µL, respectively. Initial CRP level was <10 mg/L in 45% of the participants who received antibiotics at first presentation. Conclusions: In patients with persistent fever, CRP and PCT showed higher sensitivity for bacterial infections than WBC count, applying commonly used cutoffs for normal values. A normal CRP value excluded the vast majority of severe infections and could therefore assist in deciding whether to withhold empiric antibiotics after cautious clinical assessment.